ИСТИНА

HIF (hypoxia inducible factor) prolyl hydroxylases (PHDs) belong to alpha-ketoglutarate de-pendent non-heme iron dioxygenases catalyzing proline hydroxylation in HIF. PHD2 has been reacti-vated by refolding from inclusion bodies. To optimize the refolding procedure a novel continuous spectrophotometric assay has been developed by monitoring ferrocyanide oxidation accelerated in the presence of HIF protein or peptide substrate. Comparison of the soluble enzyme with the one reacti-vated from inclusion bodies demonstrates a 4-5-fold higher specific activity of the latter (5 mol/min/vol) assayed by formation of a fluorescent adduct of ketoglutarate with o-phenylene diamine. FAD-monoxygenase domain of murine MICAL1 was expressed in E. coli and reactivated from inclusion bodies using a ferricyanide reduction assay. Despite both enzyme are unstable in the absence of DTT, the novel assays developed allow one to easily optimize refolding and purification protocols. This work was supported by Russian Foundation for Basic Research, grant 13-04-01909-а.